Inhibitors of rotamase enzyme activity effective at stimulating neuronal growth

ABSTRACT

This invention relates to the method of using neurotrophic pipecolic acid derivative compounds having an affinity for FKBP-type immunophilins as inhibitors of the enzyme activity associated with immunophilin proteins, and particularly inhibitors of peptidyl-prolyl isomerase or rotamase enzyme activity to stimulate or promote neuronal growth or regeneration.

RELATED APPLICATION

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 08/474,072 filed Jun. 7, 1995.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the method of using neurotrophic pipecolicacid derivative compounds having an affinity for FKBP-type immunophilinsas inhibitors of the enzyme activity associated with immunophilinproteins, and particularly inhibitors of peptidyl-prolyl isomerase orrotamase enzyme activity.

2. Description of the Prior Art

The term immunophilin refers to a number of proteins that serve asreceptors for the principal immunosuppressant drugs, cyclosporin A(CsA), FK506, and rapamycin. Known classes of immunophilins arecyclophilins, and FK506 binding proteins, such as FKBP. Cyclosporin Abinds to cyclophilin while FK506 and rapamycin bind to FKBP. Theseimmunophilin-drug complexes interface with a variety of intracellularsignal transduction systems, especially in the immune system and thenervous system.

Immunophilins are known to have peptidyl-prolyl isomerase (PPIase) orrotamase enzyme activity. It has been determined that rotamase activityhas a role in the catalyzation of the interconversion of the cis andtrans isomer of immunophilin proteins.

Immunophilins were originally discovered and studied in immune tissue.It was initially postulated by those skilled in the art that inhibitionof the immunophilins rotamase activity leads to the inhibition of T-cellproliferation, thereby causing the immunosuppressive action exhibited byimmunosuppressive drugs such as cyclosporin A, FK506, and rapamycin.Further study has shown that the inhibition of rotamase activity, in andof itself, is not sufficient for immunosuppressant activity. Schreiberet al. Science 1990, 250, 556-559. Instead immunosuppression appears tostem from the formulation of a complex of immunosuppressant drugs andimmunophilins. It has been shown that the immunophilin-drug complexesinteract with ternary protein targets as their mode of action. Schreiberet al., Cell 1991, 66, 807-815. In the case of FKBP-FK506 and FKBP-CsA,the drug-immunophilin complexes bind to the enzyme calcineurin,inhibiting T-cell receptor signalling leading to T-cell proliferation.Similarly, the complex of rapamycin and FKBP interacts with theRAFT1/FRAP protein and inhibits signalling from the IL-2 receptor.

Immunophilins have been found to be present at high concentrations inthe central nervous system. Immunophilins are enriched 10-50 times morein the central nervous system than in the immune system. Within neuraltissues, immunophilins appear to influence nitric oxide synthesis,neurotransmitter release, and neuronal process extension.

Nitric oxide serves several roles in the body. In the brain, nitricoxide appears to be a neurotransmitter. It is formed, from arginine, bynitric oxide synthetase which oxidizes the guanidino group of arginineforming nitric oxide and citrulline. Stimulation of theN-methyl-d-aspartate (NMDA) subtype of glutamate receptors rapidly andmarkedly activates nitric oxide synthetase and stimulates cGMPformation. Inhibition of nitric oxide synthetase with argininederivatives such as nitroarginine blocks the glutamate induced increasein cGMP levels. Nitric oxide synthetase is a calcium-calmodulinrequiring enzyme and N-methyl-d-aspartate receptor activation stimulatesnitric oxide synthetase activity because the N-methyl-d-aspartatereceptor possesses a calcium channel which is opened by glutamatestimulation, allowing calcium to rush into the cells and activate thenitric oxide synthetase.

Glutamate is a physiologic neurotransmitter. However, when released inexcess, glutamate elicits neurotoxicity via N-methyl-d-aspartatereceptors. Treatment of cerebral cortical neuronal cultures withglutamate or N-methyl-d-aspartate kills up to 90% of neurons and theseeffects are blocked by N-methyl-d-aspartate antagonist drugs. ThisN-methyl-d-aspartate neurotoxicity is thought to be a major contributorto neuronal damage following vascular stroke. Thus, there is a massiverelease of glutamate following cerebral vascular occlusion and numerousN-methyl-d-aspartate antagonists block stroke damage. Phosphorylation ofnitric oxide synthetase inhibits its catalytic activity. By enhancingnitric oxide synthetase phosphorylation, FK506 might functionallyinhibit nitric oxide formation and thus block glutamate neurotoxicity.Indeed, low concentrations of FK506 and cyclosporin A both blockN-methyl-d-aspartate neurotoxicity in cortical cultures. The mediatingrole of FKBP is evident, as rapamycin reverses the therapeutic effect ofFK506. Presumably FK506, already marketed as an immunosuppressant, couldbe clinically employed in stroke patients.

FK506 also augments the phosphorylation of growth-associated protein-43(GAP43). GAP43 is involved in neuronal process extension and itsphosphorylation appears to augment this activity. Accordingly, theeffects of FK506 rapamycin and cyclosporin in neuronal process extensionhave been examined using PC12 cells. PC12 cells are a continuous line ofneuronal-like cells which extend neurites when stimulated by nervegrowth factor (NGF).

Surprisingly, it has been found that picomolar concentrations of animmunosuppressant such as FK506 and rapamycin stimulate neurite outgrowth in PC12 cells and sensory nervous, namely dorsal root ganglioncells (DRGs). Lyons et al. proc. Natl. Acad. Sci. USA, 1994, 91,3191-3195. In whole animal experiments, FK506 has been shown tostimulate nerve regeneration following facial nerve injury and resultsin functional recovery in animals with sciatic nerve lesions.

More particularly, it has been found that drugs with a high affinity forFKBP are potent rotamase inhibitors and exhibit excellent neurotrophiceffects. Snyder et al., "Immunophilins and the Nervous System", NatureMedicine, Volume 1, No. 1, January 1995, 32-37. These findings suggestthe use of immunosuppressants in treating various peripheralneuropathies and enhancing neuronal regrowth in the central nervoussystem (CNS). Studies have demonstrated that neurodegenerative disorderssuch as Alzheimer's disease, Parkinson's disease, and amyotrophiclateral sclerosis (ALS) may occur due to the loss, or decreasedavailability, of a neurotrophic substance specific for a particularpopulation of neurons affected in the disorder.

Several neurotrophic factors effecting specific neuronal populations inthe central nervous system have been identified. For example, it hasbeen hypothesized that Alzheimer's disease results from a decrease orloss of nerve growth factor (NGF). It has thus been proposed to treatAlzheimer's patients with exogenous nerve growth factor or otherneurotrophic proteins such as brain derived growth factor, glial derivedgrowth factor, ciliary neurotrophic factor, and neurotropin-3 toincrease the survival of degenerating neuronal populations.

Clinical application of these proteins in various neurological diseasestates is hampered by difficulties in the delivery and bioavailabilityof large proteins to nervous system targets. By contrast,immunosuppressant drugs with neurotrophic activity are relatively smalland display excellent bioavailability and specificity. However, whenadministered chronically, immunosuppressants exhibit a number ofpotentially serious side effects including nephrotoxicity, such asimpairment of glomerular filtration and irreversible interstitialfibrosis (Kopp et al., 1991, J. Am. Soc. Nephrol. 1:162); neurologicaldeficits, such as involuntary tremors, or non-specific cerebral anginasuch as non-localized headaches (De Groen et al., 1987, N. Engl. J. Med.317:861); and vascular hypertension with complications resultingtherefrom (Kahan et al., 1989 N. Engl. J. Med. 321: 1725).

The present invention provides non-immunosuppressive as well asimmunosuppressive pipecolic acid derivative compounds containing smallmolecule FKBP rotamase inhibitors which are extremely potent inaugmenting neurite outgrowth, and for promoting neuronal growth, andregeneration in various neuropathological situations where neuronalrepair can be facilitated including peripheral nerve damage by physicalinjury or disease state such as diabetes, physical damage to the centralnervous system (spinal cord and brain), brain damage associated withstroke, and for the treatment of neurological disorders relating toneurodegeneration, including Parkinson's disease, Alzheimer's disease,and amyotrophic lateral sclerosis.

SUMMARY OF THE INVENTION

This invention relates to the method of using neurotrophic pipecolicacid derivative compounds having an affinity for FKBP-type immunophilinsas inhibitors of the enzyme activity associated with immunophilinproteins, and particularly inhibitors of peptidyl-prolyl isomerase orrotamase enzyme activity.

A preferred embodiment of this invention is a method of treating aneurological disorder in an animal, comprising:

administering to an animal an effective amount of a pipecolic acidderivative having an affinity for FKBP-type immunophilins to stimulategrowth of damaged peripheral nerves or to promote neuronal regeneration,wherein the FKBP-type immunophilin exhibits rotamase activity and thepipecolic acid derivative inhibits said rotamase activity of theimmunophilin.

Another preferred embodiment of this invention is a method of treating aneurological disorder in an animal, comprising:

administering to an animal an effective amount of a pipecolic acidderivative having an affinity for FKBP-type immunophilins in combinationwith an effective amount of a neurotrophic factor selected from thegroup consisting of neurotrophic growth factor, brain derived growthfactor, glial derived growth factor, cilial neurotrophic factor, andneurotropin-3, to stimulate growth of damaged peripheral nerves or topromote neuronal regeneration, wherein the FKBP-type immunophilinexhibits rotamase activity and the pipecolic acid derivative inhibitssaid rotamase activity of the immunophilin.

Another preferred embodiment of this invention is a method ofstimulating growth of damaged peripheral nerves, comprising;

administering to damaged peripheral nerves an effective amount of apipecolic acid derivative compound having an affinity for FKBP-typeimmunophilins to stimulate or promote growth of the damaged peripheralnerves, wherein the FKBP-type immunophilins exhibit rotamase activityand the pipecolic acid derivative inhibits said rotamase activity of theimmunophilin.

Another preferred embodiment of this invention is a method ofstimulating growth of damaged peripheral nerves, comprising:

administering to damaged peripheral nerves an effective amount of apipecolic acid derivative compound having an affinity for FKBP-typeimmunophilins to stimulate growth of damaged peripheral nerves, whereinthe FKBP-type immunophilin exhibit rotamase activity and the pipecolicacid derivative inhibits said rotamase activity of the immunophilin.

Another preferred embodiment of this invention is a method for promotingneuronal regeneration and growth in animals, comprising:

administering to an animal an effective amount of a pipecolic acidderivative compound having an affinity for FKBP-type immunophilins topromote neuronal regeneration, wherein the FKBP-type immunophilinsexhibit rotamase activity and the pipecolic acid derivative inhibitssaid rotamase activity of the immunophilin.

Yet another preferred embodiment of this invention is a method forpreventing neurodegeneration in an animal, comprising:

administering to an animal an effective amount of a pipecolic acidderivative having an affinity for FKBP-type immunophilins to preventneurodegeneration, wherein the FKBP-type immunophilin exhibits rotamaseactivity and the pipecolic acid derivative inhibits said rotamaseactivity of the immunophilin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A and B)

FKBP-12 and GAP-43 expression in the facial nucleus after nerve crush.In situ hybridization comparing the time course of expression of mRNA inthe facial nucleus for FKBP12 (left) and GAP-43 (right). The rightfacial nucleus is ipsilateral to the crush, and the left side is anunoperated control (FIG. 1B). In situ hybridization for FKBP-12 on anuntreated control (left) and for calcineurin Aα,β 7 days followingfacial nerve crush (right).

Experiments were replicated at least 3 times with similar results.

FIG. 2(A and B)

Localization of FKBP-12 to facial motor neurons following nerve crush.Bright-field photomicrographs of in situ hybridization for FKBP-12 inmotor neurons of the facial nucleus 7 days after crush (FIG. 2A), and inmotor neurons of control facial nucleus. (FIG. 2B).

FIG. 3(A and C)

Upregulation of FKBP-12 mRNA in lumbar spinal cord motor neurons aftersciatic nerve crush. In situ hybridization for FKBP-12 7 days aftercrush of the right sciatic nerve. Top panel (FIG. 3A) shows the responseof motor neurons in the ventral horn of lower lumbar spinal cord(indicated by the arrow). Bright field photomicrographs of correspondingmotor neuron pools are shown in the bottom panels: (FIG. 3B) left sidecontralateral to nerve crush, (FIG. 3C) right side ipsilateral to thenerve crush. This experiment was repeated 3 times with similar results.

FIG. 4

Induction of FKBP and FKBP-12 mRNA in the dorsal root ganglion 1 and 6weeks after sciatic nerve crush. Dark-field photomicrographs of sectionsthrough the L4 dorsal root ganglion ipsilateral to sciatic nerve crushprocessed for FKBP in situ hybridization are shown in the left panelsand for ³ H!FK506 autoradiography in the right panels. These resultswere replicated 3 times for each time point.

FIG. 5(A and B)

Ricin lesion of the right facial nerve. Nissl stain (bottom panel, FIG.5A) reveals extensive degeneration of motor neurons in the right facialnucleus with an accompanying glial proliferation 7 days followinginjection of ricin into the facial nerve. In situ hybridization for FKBPmRNA 7 days after ricin lesion of the facial nerve/nucleus is shown inthe top panel (FIG. 5B). This experiment was replicated 3 times withsimilar results.

FIG. 6

³ H!FK506 binding in segments of sciatic nerve 7 days following crush.The diagram illustrates the 3 mm segments of nerve taken: constrictionsindicate positions of ligatures applied at day 7 for the 6 hr collectiontime as described in the methods. The distal ligature site is 10 mmproximal to the original crush site. Anterograde transport of FKBP is124 mm/day. Data are the means±S.E.M. (n=3).

FIG. 7(A-D)

Transport of FKBP in the sciatic nerve. Dark-field photomicrographs ofsections through a control (untreated) sciatic nerve and a 7 day sciaticnerve crush site processed for FKBP-12 in situ hybridization (FIG. 7A,FIG. 7B) and for ³ H!FK-506 autoradiography (FIG. 7C, FIG. 7D). Arrowsindicate the sight of the nerve crush. This experiment was repeated 3times with similar results.

FIG. 8

Levels of ³ H!FK506 binding in PC-12 cells maintained in the presence orabsence of NGF (50 ng/ml) .n=3 for each time point. Bars representS.E.M.

FIG. 9(A-D)

Immunosuppressant mediated enhancement of neurite outgrowth in PC-12cells. Hoffman contrast photomicrographs (64) of cultures grown for 48hr in the presence of NGF with or without added FK506 or rapamycin. FIG.9A: PC-12 cells grown in 1.0 ng/ml NGF. FIG. 9B: 50 ng/ml NGF. FIG. 9C:1.0 ng/ml NGF and 100 nM FK506. FIG. 9D: 1.0 ng/ml NGF and 100 nMrapamycin. Magnification 200 X.

FIG. 10

Effects of FK506 on neurite outgrowth in PC-12 cells. Cultures weretreated with varying concentrations of NGF in the presence or absence or100 nM FK506, and neurite sprouting was measured at 48 hr. Outgrowth wasquantitated as described in Methods by counting cells with neuriticprocesses greater than 5 μm. n=4 separate experiments for each point anderror bars represent SEM.

FIG. 11

Concentration-response relationship for FK506 potentiation of neuriteoutgrowth in PC-12 cells. Cells were treated for 48 hr with 1 ng/ml NGFand varying concentrations of FK506. Neurite outgrowth response wasmeasured as described in FIG. 10 and Methods. n=4 separate experimentsfor each data point *p<0.001 Students t test.

FIG. 12

³ H!FK-506 autoradiography on dorsal root ganglion explant cultures.After 26 days of cultures with 100 ng/ml NGF the extensive processesdisplay abundant FKBP associated silver grains. Autoradiographic grainsare abolished with 1 μM unlabeled FK506.

FIG. 13(A-F)

Phase-contrast micrographs of dorsal root ganglia grown with differentsubstances. FIG. 13A: NGF 100 ng/ml, FIG. 13B: FK506 1 μM, FIG. 13C:FK506 1 μM and anti-NGF antibody, FIG. 13D: No added growth factor, FIG.13E: FK506 1 pM, FIG. 13F: FK506 1 μM. and rapamycin 1 μM. Scale bar is205 μm. NGF produces abundant axon outgrowth (FIG. 13A), as does 1 μMFK506 (FIG. 13B). The effects of FK506 are substantially decreased byreducing the concentration to 1 pM (FIG. 13E). However, neuriteoutgrowth with 1 pM FK506 is greater than in its absence (FIG. 13D).FK506 effects are also diminished by adding anti-NGF antibody toeliminate the effects of NGF produced by non-neuronal cells in thecultures. The abundant neurites that occur in large fascicles inresponse to NGF (100 ng/ml!) (FIG. 13A) or 1 μM FK506 (FIG. 13B) appearwhite, while small fascicles or individual neurites appear black.Non-neuronal cells, Schwann cells and some fibroblasts, are more evidentwith 1 pM FK506 (FIG. 13E) or anti-NGF antibody (FIG. 13C) than with 1μM FK506 (FIG. 13B). NGF produced by non-neuronal cells in the culturesresults in the limited axon outgrowth seen in cultures with no addedgrowth factors (FIG. 13D). The large number of refractile non-neuronalcells, appearing white, tend to overshadow the few neurites (FIG. 13D).Rapamycin completely inhibits axon outgrowth in the presence of FK506(FIG. 13F). Micrographs are representative of 12-30 ganglia from eachexperimental condition. Differences between all experimental groups werehighly reproducible.

FIG. 14

Effects of FK506 and rapamycin on NGF-mediated neurite extension in PC12cells. PC12 cells (passage 60) were treated with various concentrationsof NGF alone or in the presence of 100 nM FK506, 100 nM rapamycin or 100nM WAY-124,466. Neurite outgrowth was measured after 96 hours with cellsbearing processes longer than the diameter of the cells scoringpositive. n=3 separate experiments for each point and error barsrepresent S.E.M.

FIG. 15

Picomolar concentrations of (A) FK506 and (B) rapamycin and WAY-124,466potentiate neurite extension elicited by NGF (0.5 ng/ml) in PC12 cells.Low passage PC12 cells were treated for 4 days with 0.5 ng/ml NGF in thepresence of various concentrations of FK506 (□), rapamycin ( ) orWAY-124,466 ( ). Neurite expression was quantitated as described abovein FIG. 14. The levels of neurite production in the presence of 0.5ng/ml NGF (designated L) and 50 ng/ml NGF (designated H) are indicatedfor comparative purposes.

FIG. 16(A-F)

Photomicrographs of PC12 cells treated with immunophilin ligands+0.5ng/ml NGF itself or 50 ng/ml NGF.

FIG. 17(A-C)

Immunophilin ligands reduce the amount of NGF required to producemaximal neurite extension in chick sensory ganglia. Whole dorsal rootganglion explants were isolated from day 9-10 chick embryos and culturedin Matrigel-coated 12-well dishes containing L15 medium plus highglucose , with 10% fetal calf serum supplemented with 10 μM Ara Cpenicillin and streptomycin) at 37° C. in a 5% CO₂ environment. Sensoryganglia were treated with 1 ng/ml NGF, 1 ng/ml NGF plus 100 nM FK506 or100 ng/ml NGF for 48 hr, and neuronal processes were counted andphotographed.

FIG. 18

FK506, rapamycin, and WAY-124,466 potentiate NGF-dependent neuriteproduction in sensory ganglia. Explants of chick DRG were cultured asdescribed in FIG. 17 above. FK506, rapamycin and WAY-124,466 (100 nMeach plus or minus 0.1 ng/ml NGF were added to the DRG explant cultures.At 48 hrs., neurite outgrowth was quantitated and the cultures werephotographed.

FIG. 19

Photomicrograph of Example 111 promoting neurite outgrowth in Chickdorsal root ganglion cultures. The three panels show neurite outgrowthat 1 pM concentration (left panel), 100 pM concentration (center panel),and 100 nM concentration (right panel) of Example 111.

FIG. 20

Photomicrograph of Example 17 promoting neurite outgrowth in dorsal rootganglion cultures. The three panels show neurite outgrowth at 1 pMconcentration (left panel), 100 pM concentration (center panel), and 100nM concentration (right panel) of Example 17.

FIG. 21

Photomicrograph of Example 102 promoting neurite outgrowth in dorsalroot ganglion cultures. The three panels show neurite outgrowth at 1 pMconcentration (left panel), 100 pM concentration (center panel), and 100nM concentration (right panel) of Example 102.

DETAILED DESCRIPTION OF THE INVENTION

The novel neurotrophic pipecolic acid derivative compounds of thisinvention have an affinity for the FK506 binding proteins such asFKBP-12. When the neurotrophic compounds of the invention are bound toFKBP, they have been found to inhibit the prolyl- peptidyl cis-transisomerase activity, or rotamase activity of the binding protein andunexpectedly stimulate neurite growth.

The compounds of the present invention can be used in the form of saltsderived from inorganic or organic acids and bases. Included among suchacid salts are the following: acetate, adipate, alginate, aspartate,benzoate, benzenesulfonate, bisulfate butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemissulfate heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalensulfonate, nicotinate, oxalate, pamoate,pectinate, propionate, succinate, tartrate, thiocyanate, rosylate andundecanoate. Base salts include ammonium salts, alkali metal salts suchas sodium and potassium salts, alkaline earth metal salts such ascalcium and magnesium salts, salt with organic bases such asdicyclohexylamine salts, N-methyl-D-glucamine, and salts with aminoacids such as arginine, lysine, and so forth. Also, the basicnitrogen-containing groups can be quarternized with such agents as loweralkyl halides, such as methyl, ethyl, propyl, and butyl chloride,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyland diamyl sulfates, long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides, aralkyl halides like benzyland phenethyl bromides and others. Water or oil-soluble or dispersibleproducts are thereby obtained.

The neurotrophic compounds of this invention can be periodicallyadministered to a patient undergoing treatment for neurologicaldisorders or for other reasons in which it is desirable to stimulateneuronal regeneration and growth, such as in various peripheralneuropathic and neurological disorders relating to neurodegeneration.The compounds of this invention can also be administered to mammalsother than humans for treatment of various mammalian neurologicaldisorders.

The novel compounds of the present invention are potent inhibitors ofrotamase activity and possess an excellent degree of neurotrophicactivity. This activity is useful in the stimulation of damaged neurons,the promotion of neuronal regeneration, the prevention ofneurodegeneration, and in the treatment of several neurologicaldisorders known to be associated with neuronal degeneration andperipheral neuropathies. The neurological disorders that may be treatedinclude but are not limited to: trigeminal neuralgia, glossopharyngealneuralgia, Bell's Palsy, myasthenia gravis, muscular dystrophy,amyotrophic lateral sclerosis, progressive muscular atrophy, progressivebulbar inherited muscular atrophy, herniated, ruptured or prolapsedinvertebrate disk syndromes, cervical spondylosis, plexus disorders,thoracic outlet destruction syndromes, peripheral neuropathic such asthose caused by lead, dapsone, ticks, porphyria, or Guillain-Barresyndrome, Alzheimer's disease, and Parkinson's disease.

For these purposes the compounds of the present invention may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir indosage formulations containing conventional non-toxicpharmaceutically-acceptable carriers, adjuvants and vehicles. The termparenteral as used herein includes subcutaneous, intravenous,intramuscular, intraperitoneally, intrathecally, intraventricularly,intrasternal and intracranial injection or infusion techniques.

To be effective therapeutically as central nervous system targets theimmunophilin-drug complex should readily penetrate the blood-brainbarrier when peripherally administered. Compounds of this inventionwhich cannot penetrate the blood-brain barrier can be effectivelyadministered by an intraventricular route.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques know in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. Fatty acidssuch as oleic acid and its glyceride derivatives find use in thepreparation of injectables, olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant.

The compounds may be administered orally in the form of capsules ortablets, for example, or as an aqueous suspension or solution. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweeteningand/or flavoring and/or coloring agents may be added.

The compounds of this invention may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The compounds of this invention may also be administered optically,especially when the conditions addressed for treatment involve areas ororgans readily accessible by topical application, including neurologicaldisorders of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas.

For ophthalmic use, the compounds can be formulated as micronizedsuspensions in isotonic, pH adjusted sterile saline, or, preferably, assolutions is isotonic, pH adjusted sterile saline, either with orwithout a preservative such as benzylalkonium chloride. Alternativelyfor the ophthalmic uses the compounds may be formulated in an ointmentsuch as petrolatum.

For application topically to the skin, the compounds can be formulatedin a suitable ointment containing the compound suspended or dissolvedin, for example, a mixture with one or more of the following: mineraloil, liquid petrolatum, white petrolatum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the compounds can be formulated in a suitable lotion orcream containing the active compound suspended or dissolved in, forexample, a mixture of one or more of the following: mineral oil,sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearylalcohol, 2-octyldodecanol, benzyl alcohol and water.

Topical application for the lower intestinal tract an be effected in arectal suppository formulation (see above) or in a suitable enemaformulation.

Dosage levels on the order of about 0.1 mg to about 10,000 mg.of theactive ingredient compound are useful in the treatment of the aboveconditions, with preferred levels of about 0.1 mg. to about 1,000 mg.The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration.

It is understood, however, that a specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, rate of excretion, drug combination,and the severity of the particular disease being treated and form ofadministration.

The compounds can be administered with other neurotrophic agents such asneurotrophic growth factor (NGF), glial derived growth factor, brainderived growth factor, ciliary neurotrophic factor, and neurotropin-3.The dosage level of other neurotrophic drugs will depend upon thefactors previously stated and the neurotrophic effectiveness of the drugcombination.

METHODS AND PROCEDURES

Sciatic Nerve Crush

This example demonstrates high levels of FKBP in normal peripheral nerveand that these increase following nerve crush.

If FKBP were physiologically associated with neuronal process extensionin the actions of GAP-43, then one might anticipate substantial levelsof FKBP in peripheral nerve. Accordingly, we measured 3H!FK-506 bindingin rat sciatic nerve, as well as in growth cones isolated from 2-day-oldrat pups, and compared values with those of the cerebral cortex andseveral peripheral tissues.

³ H!FK-506 autoradiography was carried out as described on unfixedsections which were thawed and air dried before preincubation for 1 hrin buffer consisting of 50 mM Hepes, 2 mg/ml bovine serum albumin, 5%ethanol, and 0.02% Tween 20 pH 7.4. Sections were then exposed to 1 nM ³H!FK-506 (86.5 Ci/mMol; DuPont-NEN, Boston, Mass.) for 1 hr at roomtemperature in preincubation buffer. Non-specific binding was defined byaddition of 1 μM FK-506. Following incubation, the slides were washed4×5 min in ice cold preincubation buffer and air dried. The radiolabeledsections were then juxtaposed to tritium-sensitive film or coverslipscoated with Kodak NTB-2 emulsion.

                  TABLE 1                                                         ______________________________________                                         .sup.3 H! FK506 Binding to Sciatic Nerve and Growth Cones                    (A)  .sup.3 H! FK506 Binding in Sciatic Nerve                                                Bmax                                                           Tissue         (pmol/mg protein)                                              ______________________________________                                        Adult Rat                                                                     Sciatic Nerve  22.1                                                           Cerebral Cortex                                                                              38.0                                                           Thymus          9.5                                                           Spleen          8.0                                                           Neonatal Rat                                                                  Forebrain      25.5                                                           Growth Cones   10.2                                                           ______________________________________                                        (B)  .sup.3 H! FK506 Binding After Sciatic Nerve Crush                                     Bmax         Bmax                                                protein      fmol/5 mm segment                                                                          pmol/mg                                             ______________________________________                                        Unoperated   31.8 ± 2.1                                                                              21.2 ± 1.4                                       7-Day Crush  136.5 ± 15.7*                                                                           40.1 ± 2.0*                                      ______________________________________                                          .sup.3 H! FK506 binding was assayed as described in methods. In Table 1A     experiments were replicated three times with less than 10% variation. In      Table 1B values are presented as the mean ± S.E.M. (n = 3).                *P ≦ 0.05 Students' ttest for independent means.                  

Of all the tissues examined sciatic nerve binding levels are thehighest, somewhat higher than those of the cerebral cortex and about10×higher than levels in the thymus and spleen, which contain FKBPassociated with lymphocytes. See Table 1A.

Evidence for a role of FKBP in nerve regeneration comes from experimentsin which we crushed the sciatic nerve of adult rats and 7 days latermeasured ³ H!FK506 binding in a 5 mm segment immediately proximal to thenerve crush.

Sprague-Dawley rats (175-200 g) were anesthetized with a mixture ofRompun (12 mk/kg) Ketamine (30 mg/kg). Using aseptic techniques, thefacial nerve was crushed with jewelers forceps 2×30 sec 2 mm distal toits exit from the stylomastoid foramen. Identical procedures were usedto crush the sciatic nerve at the level of the mid-thigh.

Total binding in the segment proximal to the crush was quadrupledcompared to control values. Since total protein is substantiallyaugmented in the proximal segment, ³ H!FK-506 binding per mg protein isonly doubled in the proximal segment.

Facial Nerve Crush

This example demonstrates that facial nerve lesions augment thecoincident expression of FKBP and GAP-43.

Following the crush of the facial nerve, mRNA levels of GAP-43 increasein the facial nerve nucleus. Utilizing in situ hybridization, weexamined mRNA levels of FKBP, GAP-43 and calcineurin following facialnerve crush.

Rats were perfused transcardially with 150-200 ml ice coldphosphate-buffered saline (PBS) (O.1M, pH 7.4). Tissues were removed andimmediately frozen in isopentane (-80° C.). Cryostat sections 18 μmthick) were cut and thaw mounted on gelatin coated slides.

In situ hybridization was performed as previously described, usingantisense oligonucleotide probes end labeled with ³⁵ S!dATP. For FKBP,three separate oligonucleotides complementary to the following regionsof the cloned cDNA disclosed by Maki, et al. (1990) Proc. Natl.Acad.-Sci. USA 87, 5440-5443, and Standaert, R. F., et al. (1990) Nature346, 671-674 and incorporated herein by reference, were used: 70-114,214-258, 441-485. For GAP-43, three separate antisense oligonucleotidescomplementary to nucleotides 961-1008, 1081-1128, 1201-1248 of thecloned cDNA disclosed by Rosenthal, A., et al. (187) EMBO J. 6,3641-3646 and incorporated herein by reference were used. Forcalcineurin Aα antisense oligonucleotides complementary to thenucleotides 1363-1410 and 1711-1758, disclosed by Ito et al. (1989)Biochem. Biophys. Res. Commun. 163, 1492-1497 and incorporated herein byreference, and for calcineurin Aβ 1339-1386 and 1569-1616 disclosed byKuno, T., et al. (1989) Biochem. Biophys. Res. Commun. 165, 1352-1358and incorporated herein by reference, were used. Sections were thawedand allowed to dry, then fixed for 5 min in 4% freshly depolymerizedparaformaldehyde in PBS. Following two rinses in PBS, sections wereacetylated with 0.25% acetic anhydride in 0.1M triethanolamine 0.5% NaCl(pH 8.0), and then dehydrated in graded alcohols, defatted in chloroformfor 5 min, rehydrated to 95% ethanol and allowed to air dry.Hybridization was performed overnight at 37° C. in buffer containing 50%deionized formamide, 10% dextran sulfate, 4×SSC, 1×Denhardt's solution,20 mM phosphate buffer, 0.1 mg/ml salmon sperm DNA, 0.1 mg/ml yeasttransfer RNA, 10 mM dithiothreitol, 2.0% betamercaptoethanol (BMD), 1.0mM EDTA and labelled probe (2,000,000 dpm/section). Followinghybridization, sections were rinsed in 1×SSC, 1.0% BME for 15 min atroom temperature, then twice for 10 min at 55° C. air dried and placedon film or dipped in Kodak NTB-2 emulsion.

Striking enhancement of FKBP and GAP-43 expression is observed, while nochanges are evident in calcineurin expression. As early as 24 hrfollowing facial nerve crush FKBP expression is increased with peaklevels evident a 1-2 weeks, while mRNA concentrations diminishsubstantially at 3 weeks. Examination under higher magnification revealsthat the increased levels of silver grains for FKBP mRNA are confined toneuronal cell bodies (FIG. 2). Northern blot analysis of the dissectedfacial nucleus confirms the increased levels of FKBP specific mRNA.GAP-43 mRNA levels follow a time course closely similar to those ofFKBP. By contrast, no changes in calcineurin expression are detected atany of the time points examined.

Total cellular RNA from the dissected facial nucleus was isolated.Samples of 10 or 20 ug total RNA were electrophoresed through a 1%agarose, 2.0% formaldehyde gel and transferred to a nylon membrane in 10nM NaOH. cDNA probes to FKBP labeled with ³⁵ !dCTP to a specificactivity of 1×10⁹ cpm/ug by random priming were hybridized overnight at42° C. in buffer consisting of 50% formamide, 2×SSPE, 7% SDS, 0.5%Blotto and 100 ug/ml salmon sperm DNA. The blots were washed for 20 minat room temperature, and 2×15 min at 65° C. in 0.15×SSC, 0.15% SDS andthen exposed to film for 48096 hrs.

On the unlesioned side a modest increase in silver grains compared tocontrol sections are observed. This is consistent with findings thatcontralateral neurons also respond to axotomy.

Following facial nerve crush, rats develop a facial nerve palsy, whichis evident by the lack of whisker movement with functional recovery at 3weeks coincident with the completion of nerve regeneration. In our ratswe also observed the loss of whisker movement following nerve crush witha return of function at 3 weeks. Thus, the time course of increasedexpression of GAP-43 and FKBP correlates with the process of nerveregeneration.

Sciatic Nerve Regeneration

This example demonstrates alterations in FKBP and GAP-43 associated withsciatic nerve regeneration.

Following sciatic nerve lesions GAP-43 mRNA levels are enhanced in bothspinal cord motor neurons and in dorsal root ganglia neuronal cells. Inrats subjected to sciatic nerve crush, we observed a strikingenhancement in mRNA levels for FKBP in motor neurons at L-4, 5 (FIG. 3)and in dorsal root ganglia neuronal cells coincident with the reportedenhancement of GAP-43 expression (FIG. 4). At high magnification weobserved the FKMB mRNS silver grains localized to neuronal cell bodies(FIG. 3). We monitored FKBP protein levels by autoradiography of ³H!FK-506 binding under conditions in which it binds selectively to FKBP(FIG. 4). Increased FKBP is detected in the primary sensory neurons inthe dorsal root ganglia, though no increases are evident in motorneuronal cells following sciatic nerve crush.

The association of augmented FKBP expression with regenerationselectively is further supported by experiments with ricin. Wheninjected into peripheral nerves ricin is transported back into the cellbody which is destroyed without associated nerve regeneration. Weinjected 0.5 ug ricin (RCA 60, Sigma, St. Louis, Mo.) into the facialnerve at the same site where crushes had been performed in otherexperiments according to the method of Streit and Kreutzbnerg in 0.5 ulPBS and 0.1% Fast Green. Streit et al., (1988) J. Comp. Neurol. 268,248-263.

We conducted in situ hybridization localization studies for FKBP mRNA at2, 4 and 7 days following ricin treatment (FIG. 5). No increase in FKBPmRNA is observed following ricin treatment. Gliosis occurs bothfollowing ricin treatment and nerve crush. The failure of FKBP mRNA toincrease following ricin treatment fits with the selective neuronallocalization of FKBP mRNA in the facial nucleus.

FKBP Transport in the Sciatic Nerve

This example demonstrates that FKBP is rapidly transported in thesciatic nerve.

The failure of FKBP protein to increase in motor neurons followingsciatic nerve crush despite the increase in FKBP mRNA suggests that theprotein is rapidly transported out of the cell body into nerveprocesses. This fits with out earlier observations that FKBP mRNA isconcentrated in granule cells of the cerebellum which contain low levelsof FKBP protein, while FKBP protein levels are highly concentrated inthe molecular layer in the cerebellum associated with the parallelfibers arising from granule cells. To examine for possible transport ofFKBP, we crushed the sciatic nerve and 7 days later applied ligatures 10and 20 mm proximal to the crush. Six hr following ligature, we monitored³ H!FK-506 binding in 3 mm segments spanning the area of the ligatures.(FIG. 6).

For axon transport experiments, classic ligature techniques were usedfollowing the methods of Tetzlaff et al. One week following sciaticnerve crush two collection ligatures (510 sutures) were placed on thenerve approximately 10 mm apart with the distal most ligature positioned10 mm proximal to the initial crush site. Six hours later, 5-3 mmsegments of the nerve were collected from regions proximal to, distalto, and between the collection ligatures as illustrated in FIG. 5. Thenerve segments were prepared for ³ H!FK-506 binding assays byhomogenizing in 10 volumes of 50 mM Tris-HC1,pH 7.4 Homogenates werecentrifuged at 15,000×g for 20 min at 4° C., and supernatants werecollected and assayed for total protein concentration using theCoomassie blue dye binding assay (Pearce). ³ H!FK-506 binding wascarried out as described (4) on aliquots containing 2 ug of totalsoluble protein in a final volume of 0.4 ml assay buffer consisting of50 mM Tris-HC1, pH 7.4, 2 mg/ml bovine serum albumin, 250 pM ³ H!FK-506,and varying concentrations of unlabeled FK-506. Following incubation at25° C. for 60 min, 0.35 ml was layered over a 0.8 ml column of LH-20Sephadex (Pharmacia LKB) and washed with 0.4 ml of assay buffer. Theeluates were collected and counted in a scintillation counter.

Results are shown in FIG. 5. ³ H!FK-506 binding levels are highest inthe segment just proximal to the ligature 20 cm from the crush, beingalmost quadruple levels in the other segments. Based on the levels of ³H!FK-506 binding in segments A-D, we calculated the rate of anterogradetransport for FKBP. This rate of 240 mm per day is essentially the sameas transport rates for GAP-43 representing the most rapid transportrates for neuronal proteins.

To visualize the accumulation of FKBP following nerve crush, we applieda loose ligature to mark the site of crush of the sciatic nerve andconducted in situ hybridization for FKBP mRNA as well autoradiographyfor ³ H!FK-506 binding (FIG. 7). Most FKBP mRNA and ³ H!FK-506 bindingaccumulate immediately proximal to the crush. These levels areconsiderably higher than in control uncrushed sciatic nerve. Examinationof the in situ hybridization an autoradiography preparations at highmagnification reveals silver grains associated with neuronal fibers.There are also silver grains localized to cells whose identity we couldnot determine definitively, so that they may be Schwann cells,macrophages or fibroblasts.

FKBP in PC12 Cells

This example demonstrates that PC-12 cells contain FKBP and that FKBPlevels are enhanced by nerve growth factor. We examined PC-12 cells forthe presence of FKBP by monitoring the binding of ³ H!FK-506 to cellsunder basal conditions and following treatment with nerve growth factor(NGF).

Levels of FKBP in PC-12 cells were obtained from Scatchard analysis of ³H!FK-506 binding curves. Cultures were scraped from the culture wellsand homogenized in 10 volumes of 50 mM Tris-HC1,pH 7.4, 1 mM EDTA, 100μg/ml phenylmethylsulfonylfluoride and centrifuged at 40,000×g for 20min at 4° C. Protein was determined by the Coomassie blue dye bindingassay using bovine serum albumin as a standard. Binding of 250 pM ³H!dihydro FK506 (86.5 Ci/mmol, DuPont/NEN) was assessed for samplescontaining 5 μg soluble protein in a final volume of 0.4 ml assay buffercontaining 50 mM Tris-HC1, pH 7.4, 2 mg/ml BSA and varyingconcentrations of unlabeled FK506. After 60 min incubation at 25° C.,0.35 ml was layered over a 0.8 ml column of LH-20 Sephadex (PharmaciaLKB), pre-equilibrated with assay buffer. The column was further washedwith 0.4 ml of assay buffer, the eluates collected, mixed with Formula963 (DuPont/NEN) and counted in a Beckman scintillation counter.Specific binding was determined by subtracting binding obtained in thepresence of 1 μM unlabeled FK506 from total ³ H!FK506 bound.

Results are shown in FIG. 8. ³ H!FK506 binds saturably to untreatedPC-12 cell homogenates. In typical experiments about 1,000 cpm are boundwhile nonspecific binding in the presence of 1 μM FK506 is about 150cpm. Fifty percent inhibition of ³ H!FK506 binding occurs with 1-2 nMFK506 indicating that the binding sites correspond to authentic FKBP. ³H!FK506 binding increases markedly following NFG treatment. Significantincreases are evident by 10-15 hr. Binding triples by 20 hr and a modestfurther increase is evident at 100 hr.

Increased Neurite Extension in PC12 Cells

This example demonstrates that FK506 and rapamycin increase neuriteextension in PC-12 cells.

PC-12 cells were maintained at 37° C., 5% CO₂, in Dulbecco's modifiedEagle medium (DMEM) supplemented with 10% heat-inactivated horse serumand 5% heat-inactivated fetal bovine serum. For differentiation in NGF,cells were plated at 1×10⁵ in 35 mm culture wells coated with rat tailcollagen at 5 μg/cm², and allowed to attach before replacing the mediawith DMEM supplemented with 2% fetal horse serum, NGF and/or FK506 ofrapamycin. For quantitation of neurite outgrowth, random photographswere made (3-4 per well), and process bearing neurons were counted withprocesses being greater than 5 μm. Experimental conditions were unknownby the photographer and cell counter. Four separate experiments wereperformed in duplicate for each data point presented. Neurites wereidentified and counted from approximately 100 cells per photograph.Thus, neurites from 1200-1600 cells were counted per data point.

As observed, NGF potently stimulates neurite outgrowth with half-maximalstimulation at 1 ng/ml and maximal augmentation at about 50-100 ng/ml(FIGS. 9, 10). FK506 (100 nM) markedly augments the effect of NGF byincreasing sensitivity to NGF. Thus, FK506 reduces by 20-50 fold the NGFconcentration needed to elicit maximal outgrowth. Half maximal outgrowthin the absence of FK506 occurs at 5 ng/ml NGF and in the presence ofFK506 at 0.1 ng/ml NGF. At maximal concentrations of NGF (10-100 ng/ml),FK506 fails to produce additional neurite outgrowth.

FK506 is extremely potent in its neurotrophic effects. In the presenceof a submaximal concentration of NGF (1 ng/ml) FK506 at 1 nM elicits thesame maximal outgrowth observed with 50 ng/ml NGF (FIG. 11). Halfmaximal effects of FK506 occur at approximately 100 pM. In the absenceof NGF, FK506 fails to elicit neurite outgrowth (FIG. 10).

Rapamycin is a potent immunosuppressant which is not thought to actthrough calcineurin but which may influence other phosphorylationcascades. Rapamycin potently blocks actions of FK506 that occur throughFKBP and calcineurin presumably by acting as an FK506 antagonist atFKBP. Rapamycin (1 μM) fails to block the neurotrophic actions of FK506.Instead, rapamycin is itself neurotrophic providing major neuriteoutgrowth at 1 nM. Rapamycin and FK506 seem to be acting via differentmechanisms. Thus, rapamycin augments the number of processes as well astheir length, while FK506 primarily increases neurite length. Moreover,effects of FK506 and rapamycin appear to be additive.

Dorsal Root Ganglia

This example demonstrates that FK506 is neurotrophic for sensoryganglia. We examined the action of FK506 on primary cultures of dorsalroot ganglia from rats at embryonic day 16.

Stage E16 embryos were removed from pregnant Sprague-Dawley rats and thedorsal root ganglia dissected. Whole ganglia explants were cultured incollagen-coated 35 mm dishes (Falcon) using N2 medium (Dulbecco'sModified Eagle medium and Ham's F12 medium mixed 1:1 and supplementedwith progesterone, selenium, insulin, putrescine, glucose, andpenicillin-streptomycin) at 37° C. in a 15% CO₂ environment. Sensoryganglia were treated with various concentrations of NGF and/or FK506 orrapamycin or anti-NGF antibody. Ganglia were observed every 2-3 daysunder phase-contrasting using an Olympus IMT-2 inverted microscope, andmeasurements of axon length were made. The axonal field of each ganglionwas divided into four quadrants, and the length of the longest axons ineach quadrant was measured in microns using an eye-piece micrometer. Theaverage of these measurements was taken as the axon length for theganglion.

For ³ H!FK506 autoradiography, dorsal root ganglia cultures were grownon chamber slides coated with collagen, 5 μg/cm². Cultures were fixed onthe slide with ice cold 4.0% freshly depolymerized paraformaldehyde in0.1M sodium phosphate buffer, pH 7.4, for 1 hr, then washed two timeswith phosphate buffered saline. Fixed cultures were labeled with ³H!FK506 by pre-incubating the slides in a buffer consisting of 50 mMHepes, 2 mg/ml bovine serum albumin, 0.02% Tween-20 pH 7.4. This wasfollowed by incubation in the same assay buffer containing 1 nM ³H!FK506. Non-specific binding was determined by adding 1 μM unlabeledFK506. The slides were then rinsed 4×5 min prior to drying, andjuxtaposed to tritium-sensitive film for 10 days.

Autoradiography of ³ H!FK506 binding sites reveals substantial levels ofFKBP associated silver grains in these ganglia (FIG. 12). At 1 μMunlabeled FK506, autoradiographic grains are abolished indicating thespecificity of binding. As reported previously, NGF (100 ng/ml) markedlyincreases the number and length of ganglia processes (FIG. 13). FK506 (1μM) alone produces a similar neurotrophic effect, while as little as 1nM FK506 produces a noticeable increase in growth. Rapamycin (1 μM)which acts as an FK506 antagonist, completely blocks the effects ofFK506 (1 μM), thus the action of FK506 displays a drug specificitycharacteristic of FKBP.

Whereas FK506 fails to stimulate neurite outgrowth in PC-12 cells in theabsence of added NGF, in sensory ganglia FK506 alone is neurotrophic.Schwann cells in the ganglia can fabricate NGF, and the production ofNGF by Schwann cells is regulated by a protein phosphorylation event. Toascertain whether the actions of FK506 alone involve potentiation ofendogenous NGF, we examined the influence of antibodies to NGF (FIG.13). Anti-NGF markedly reduces the neurotrophic effects of FK506 (1 μM).The anti-NGF is not acting in a toxic fashion as we observe nomorphologic evidence of toxicity in the cells exposed to anti-NGF in thepresence or absence of added NGF.

FK506 is extremely potent in stimulating neurite outgrowth. As little as1 pM FK506 produces detectable augmentation. Progressively greateroutgrowth occurs at 0.1 and 10 nM FK506 (data not shown), while maximaloutgrowth requires 1 μM FK506.

The time course of neurite outgrowth is similar at all concentrations ofNGF and FK506. Some outgrowth is evidence by 1 day, while growth beginsto plateau at about 5-6 days.

FK506 neurotrophic effects involve FKBP (FK506 binding protein) insensory ganglia since the effects of FK506 are reversed by lowconcentrations of rapamycin, a known antagonist of FK506 at FKBP. Thefailure of rapamycin to block FK506 effects in PC-12 cells probablyreflects the separate stimulatory effects of rapamycin. Mechanisms forrapamycin stimulation of neurite outgrowth in PC-12 cells are notimmediately evident. Its immunosuppressant actions are thought toinvolve different mechanisms than those of FK506. Rapamycin can inhibitS6 kinase which phosphorylates the S6 ribosomal subunit. Rapamycin alsoinhibits phosphatidylinositol-3-kinase.

Protein kinase C (PKC) -mediated phosphorylation has been implicated inprocess outgrowth during neuronal regeneration. Other evidence suggestsinhibitory effects of PKC in neuronal process extension.

GAP43 is a prominent calcineurin substrate highly concentrated inneurites and its phosphorylation is regulated by FKBP. GAP43 may not bedirectly involved in neurite extension, as PC-12 cell lines with lowlevels of GAP43 display normal neurite outgrowth. However, GAP43 and itsphosphorylation may be involved in targeting neurites, as levels ofphosphorylated GAP43 are increased when neurites approach their targets.Phosphorylation of GAP-43 may also influence mobilization of Ca² thatregulates neurite extension. Phosphorylated GAP-43 inhibits phosphatidylinositol bis-phosphate formation, which should diminish level ofinositol 1,4,5-triphosphate and associated Ca²⁺ release. In addition,phosphorylation of GAP-43 decreases its affinity for calmodulin with theresultant free calmodulin available to bind Ca²⁺.

Immunophilins may act at sites besides calcineurin which affect Ca²⁺that regulates neurite outgrowth. FKBP binds to the ryanodine receptor,which is a CA²⁺ release channel. In skeletal muscle sarcoplasmicreticulum FK506 dissociates FKBP from the ryanodine receptor tofacilitate the Ca²⁺ induced Ca²⁺ release mechanism. In addition, FK506acts at other sites including FKBP25 steroid receptors and otherunidentified targets such as those related to FKBP13. Thus otherpotential mechanisms may play some role in neurite extension.

Non-Immunosuppressive and Immunosuppressive Ligands of ImmunophilinsStimulate Neurite Outgrowth in PC-12 Cells

In the present study we have examined in detail influences of a numberof ligands of the immunophilins upon neurite extension in PC-12 cellsand in intact chick sensory ganglia. We report thatnon-immunosuppressive as well as immunosuppressive ligands are extremelypotent in augmenting neurite outgrowth in both PC-12 cells and sensoryganglia.

In our earlier study we found that immunosuppressants stimulate neuriteoutgrowth in PC-12 cells by increasing the potency of nerve growthfactor (NGF) about 10-fold (Lyons et. al., 1994). In the absence ofadded NGF no neurotrophic effects are observed. In the present study weevaluated effects of the immunosuppressant drugs FK506 and rapamycin onPC-12 neurite outgrowth in the presence of 0.1-100 ng/ml of NGF.

In the absence of added NGF, none of the drugs stimulate neuriteoutgrowth. At 0.1 ng/ml NGF alone produces a small increase in neuriteextension only about 15% of maximal effects that are evident at 50 ng/ml(FIG. 14). Rapamycin stimulates neurite outgrowth to a greater extentthan the other drugs, with a 3-4 fold stimulation at 0.1-0.5 ng/ml NGF.The extent of augmentation elicited by rapamycin decreases with higherconcentration of NGF and is not statistically significant at 5-50 ng/mlNGF. FK506 also is neurotrophic with effects most apparent at lower NGFconcentrations and a maximal 2.5-fold enhancement of neurite outgrowthevident at 0.5 ng/ml NGF.

There are three principal structural classes of immunosuppressant drugsrelated in structure to cyclosporin A, FK506, and rapamycin. ThoughFK506 and cyclosporin A bind to distinct immunophilin proteins, theyboth act as immunosuppressants by inhibiting calcineurin. Rapamycin bindwith very high affinity to FKBP-12, but the drug-immunophilin complexdoes not in turn bind to calcineurin. Instead, immunosuppressant actionsresult from the rapamycin-FKBP-12 complex binding to a recentlyidentified and cloned protein designated RAFT-1 (rapamycin and FK506target) and also designated FRAP (Sabatini and Snyder, 1994; Brown et.al., 1994; Chen et. al., 1994). Because rapamycin binds potently toFKBP-12 but does not inhibit calcineurin, it can serve as an antagonistto FK506. There exist non-immunosuppressive derivatives of rapamycin.One of these, WAY-124,466, a triene derivative of rapamycin, binds withhigh affinity to FKBP-12 and inhibits rotamase activity, but is devoidof immunosuppressant actions. Cyclosporin A is a large cyclicundecapeptide. The mere addition of a methyl group to an alanine at the6 position results in an agent that does not inhibit calcineurin andlacks immunosuppressive effects, though it inhibits the rotamaseactivity of cyclophilin to a similar extent as cyclosporin A (Me CsAref).

To ascertain whether immunosuppressant activity is required forneurotrophic actions, we compared the neurotrophic effects of FK506,rapamycin and cyclosporin A with non-immunosuppressant WAY-124,466,evaluating a wide range of concentrations on PC-12 cells (FIGS. 15, 16).All studies were done in the presence of 0.5 ng/ml NGF. As observedpreviously, FK506 very potently stimulates neurite extension withhalf-maximal stimulation at 0.5 nM and maximal effects at 5-100 nM.

Rapamycin is the most potent agent examined and produces the greatestmaximal level of neurite extension. In repeated experiments 50% ofmaximal extension is evident at about 0.2-0.4 nM while maximal effectsare evident at about 10-100 nM. Maximal neurite extension with rapamycinis comparable to maximal effects of 50 ng/ml NGF. WAY-124466 also isneurotrophic, but is less potent and produces a lesser maximal effectthan rapamycin. Half-maximal stimulation with WAY-124,466 occurs atabout 10 nM and maximal effects occur at 100-1,000 nM. Thus, rapamycinis about 100-fold more potent than WAY-124,466, resembling its 40-foldgreater potency in binding to FKBP-12 (Table II).

Cyclosporin A is substantially less potent than FK506 or rapamycin instimulating neurite outgrowth, corresponding to its substantially lesserpotency in inhibiting rotamase activity. Fifty percent maximalstimulation of neurite outgrowth with cyclosporin A is evident at 50 nMwith maximal effects at 100 nM and a decrease in neurite outgrowth athigher concentrations of cyclosporin A. Maximal stimulation withcyclosporin A is about 60% of effects of 50 ng/ml NGF.

The general pattern of process extension is similar with the variousimmunophilin ligands and with NGF. At concentrations that elicit 50% ofmaximal effects, NGF (1-5 ng/ml) 40-50% of cells extend processes atleast as long as the cell body while 15% extent longer processes, up to3-5 times the length of the cell body. The pattern is fairly similarwith the various drugs examined. Rapamycin and WAY-124,466 tend toresult in a greater number of processes per cell than FK506. CyclosporinA tends to be intermediate in terms of numbers or processes.

Nerve Extension Elicited in Chick Dorsal Root Ganglia byNon-Immunosuppressive and Immunosuppressive Ligands of Immunophilins

In our previous study, we observed neurotrophic effects ofimmunosuppressant drugs in explants of rat dorsal root ganglia withsignificant augmentation in nerve outgrowth observed with FK506concentrations as low as 1 picomolar (Lyons et. al., 1994). In the ratganglia neurotrophic effects were observed with FK506 even in theabsence of NGF. In the present study we have employed explants of chickdorsal root ganglia, which are easier to employ in studies of nerveoutgrowth. In the absence of added NGF, we observe minimal effects ofimmunophilin ligand drugs. The chick cells are more sensitive to NGFthan PC-12 cells so that we employ 0.1 ng/ml NGF to produce minimalneurite outgrowth and to demonstrate neurotrophic actions ofimmunophilin ligands (FIG. 17,18).

Dorsal root ganglion were dissected from chick embryos of ten daygestation. Whole ganglion explants were cultured on thin layerMatrigel-coated 12 well plates with Liebovitz L15 plus high glucosemedia supplemented with 2 mM glutamine and 10% fetal calf serum, andalso containing 10 μM cytosine β-D arabinofuranoside (Ara C) at 37° C.in an environment containing 5% CO₂. Twenty-four hours later, the DRG'swere treated with various concentrations of nerve growth factor,immunophilin ligands or combinations of NGF plus drugs. Forty-eighthours after drug treatment, the ganglia were visualized under phasecontrast or Hoffman Modulation contrast with a Zeiss Axiovert invertedmicroscope. Photomicrographs of the explants were made, and neuriteoutgrowth was quantitated. Neurites longer than the DRG diameter werecounted as positive, with total number of neurites quantitated per eachexperimental condition. Three to four DRGs are cultured per well, andeach treatment was performed in duplicate.

The relative potencies of the various immunophilin ligands instimulating nerve outgrowth in the ganglia are similar to their relativepotencies in PC-12 cells. Thus, rapamycin is the most potent agent withan EC₅₀ of 1 nM , 10-fold more potent than WAY-124,466, while FK506displays an EC₅₀ of 1-2 nM.

The maximal increase in the number of processes, their length andbranching is quite similar at maximally effective contractions of theimmunophilin ligands and of NGF (100 ng/ml). With progressivelyincreasing concentrations of the various drugs, one observes a largernumber of processes, more extensive branching and a greater length ofindividual processes.

We evaluated the potencies of drugs in binding to FKBP-12 by examininginhibition of ³ H-FK506 binding to recombinant FKBP-12. There is astriking parallel between affinities of drugs for FKBP-12 and theirpotencies in stimulating neurite outgrowth and inhibiting rotamaseactivity. Clearly, stimulation of nerve outgrowth is unrelated tocalcineurin inhibition. Calcineurin inhibition fits well withimmunosuppressant actions, WAY-124,466 is not immunosuppressive andfails to inhibit calcineurin. Rapamycin is a potent immunosuppressant,but the rapamycin-FKBP-12 complex binds to RAFT-1 to initiateimmunosuppressive processes (Sabatini and Snyder, 1994; Snyder andSabatini, 1995). The results are set forth in Table 2.

                  TABLE 2                                                         ______________________________________                                        IMMUNOPHILIN LIGAND NEUROTROPHISM PARALLELS                                   INHIBITION OF ROTAMASE, NOT CALCINEURIN                                                                       ROTAMASE                                                                      NEURITE                                       FK506-    .sup.3 H!                                                                    CALCINEURIN                                                                              OUTGROWTH                                                 DRUG     FKBP12 (IC.sub.50)                                                                       INHIBITION  (K.sub.i)                                                                            (ED.sub.50)                            ______________________________________                                        FK506     0.6 nM    YES         0.4  nM  0.5 nM                               Rapamycin                                                                               0.5 nM    NO          0.2  nM  0.5 nM                               WAY-     10.0 nM    NO          12.0 nM  IO  nM                               124466                                                                        Cyclosporin                                                                            None       YES         20   nM  50  nM                               A (CsA)                                                                       ______________________________________                                    

We compared the ability of non-immunosuppressive immunophilin ligands topromote neurite outgrowth in chick dorsal root ganglion explant cultures(Table 3). Each of these compounds is incapable of inhibitingcalcineurin, but they interact with the immunophilin FKBP-12 to inhibitits rotamase activity with the various inhibitory constants listed inTable 3. The ability of these compounds to promote neurite outgrowth inthe DRG's correlates well with their ability to inhibit the rotamaseactivity of FKBP-12.

                  TABLE 3                                                         ______________________________________                                        IMMUNOPHILIN LIGAND NEUROTROPHISM PARALLELS                                   INHIBITION OF ROTAMASE, NOT CALCINEURIN                                                                             Neurite                                 FK506-    .sup.3 H!                                                                    Calcineurin                                                                              Rotamase   Outgrowth                                      Drug     FKBP12 (IC.sub.50)                                                                       Inhibition (K.sub.i)                                                                            (ED.sub.50)                             ______________________________________                                        Example 12                                                                             8 μM    NO         250 nM 300 nM                                  Example 13                                                                             4 μM    NO          25 nM 80 nM                                   ______________________________________                                    

The very close correlation between the potencies of drugs in binding toimmunophilins, inhibiting their rotamase activity and stimulatingneurite outgrowth implies that inhibition of rotamase activity isresponsible for neurotrophic effects of the drugs. The extraordinarilyhigh potency of the drugs in stimulating neurite outgrowth and inbinding to immunophilins makes it most unlikely that any other targetcould account for the neurotrophic effects. It is conceivable that abiological activity of immunophilins other than rotamase could beinfluenced by the drugs to mediate neurotrophic actions. However, nosuch activity has yet been reported.

Because of the extraordinary potency of the drugs and the closecorrelation between rotamase inhibition and neurotrophic actions, weconclude that rotamase inhibition is likely involved in neurotrophiceffects. A number of proteins have been reported as substrates for therotamase activity of immunophilins including collagen (Steinmann et.al., 1991) and transferrin (Lodish and King, 1991). Recently highlypurified preparations of ryanodine receptor and the IP-3 receptor,prominent intracellular calcium channels have been reported to exist ina complex with FKBP-12. Dissociation of FKBP-12 from these complexescauses the calcium channel to become "leaky" (Cameron et. al., 1995)Calcium fluxes are involved in neurite extension so that the IP-3receptor and the ryanodine receptor might be involved in theneurotrophic effects of drugs. Since the drugs bind to the same site onFKBP-12 as the IP-3 receptor or the ryanodine receptor, one would haveto postulate that the drugs displace the channels from FKBP-12. Nointeraction between these calcium channels in cyclophilin has beenreported so that this model would not explain the neurotrophic actionsof cyclosporin A.

The neurotrophic actions of the drugs studied here are exerted atextremely low concentrations indicating potencies comparable to those ofneurotrophic proteins such as brain derived growth factor, neurotropin-3and neurotrophic growth factor.

The following examples are illustrative of preferred embodiments of theinvention and are not to be construed as limiting the invention thereto.All polymer molecular weights are mean average molecular weights. Allpercentages are based on the percent by weight of the final deliverysystem or formulation prepared unless otherwise indicated and all totalsequal 100% by weight.

Illustrative pipecolic acid derivative compounds which can be used forthe purposes of this invention include:

EXAMPLE 1 ##STR1##

This exemplary pipecolic acid derivative compound is disclosed by Ocainet al., Biochemical and Biophysical Research Communications, Vol. 192,No. 3, 1993. The compound was synthesized at Wyeth-Ayerst by Dr. PhilHughes by reaction of 4-phenyl-1,2,4-triazoline-3,5-dione withrapamycin.

EXAMPLE 2 ##STR2##

This pipecolic acid derivative compound is disclosed by Chakraborty etal., Chemistry and Biology, March 1995, 2:157-161.

EXAMPLES 3-5 ##STR3##

Exemplary pipecolic acid derivative compounds are disclosed by Ikeda etal., J. Am. Chem. Soc. 1994, 116, 1431-4144, and are incorporated hereinby reference.

EXAMPLES 6-9

Exemplary pipecolic acid derivative compounds are disclosed by Wang etal., Bioorganic and Medicinal Chemistry Letters, Vol. 4, No. 9, pp.1161-1166, 1994, particularly compounds 2a-2d and are incorporatedherein by reference.

EXAMPLE 10 ##STR4##

This exemplary pipecolic acid derivative, compound 10, is disclosed byBirkenshaw et al., Bioorganic & Medicinal Chemistry Letters, Vol. 4, No.21, pp. 2501-2506, 1994, and is incorporated herein by reference.

EXAMPLES 11-21

Exemplary pipecolic acid derivative compounds are disclosed by Holt etal., J. Am. Chem. Soc., 1993, 115, 9925-9938, particularly compounds4-14, and are incorporated herein by reference.

EXAMPLES 22-30

Exemplary pipecolic acid derivative compounds are disclosed by Cafferyet al., Bioorganic & Medicinal Chemistry Letters, Vol. 4, No. 21, pp.2507-2510, 1994, and are incorporated herein by reference.

EXAMPLE 31 ##STR5##

This exemplary pipecolic acid derivative, compound 31, is disclosed byTeague et al., Bioorganic & Medicinal Chemistry Letters, Vol. 3, No. 10,pp. 1947-1950, 1993 and is incorporated herein by reference,

EXAMPLES 32-34

Exemplary pipecolic acid derivative compounds are disclosed by Yamashitaet al., Bioorganic & Medicinal Chemistry Letters, Vol. 4., No. 2, pp.325-328, 1994, particularly, compounds 11, 12, and 19, and areincorporated herein by reference.

EXAMPLE 35-55

Exemplary pipecolic acid derivatives are disclosed by Holt et al.,Bioorganic & Medicinal Chemistry Letters, Vol. 4, No. 2, pp. 315-320,1994, particularly, compounds 3-21, and 23-24, and are incorporatedherein by reference.

EXAMPLES 56-68

Exemplary pipecolic acid derivative compounds are disclosed by Holt etal., Bioorganic & Medicinal Chemistry Letters, Vol. 3, No. 10, pp.1977-1980, 1993, particularly compounds 3-15 and are incorporated byreference herein.

EXAMPLES 69-83

Exemplary compounds of the present invention are disclosed by Hauske etal., J. Med. Chem. 1992, 35, 4284-4296, particularly compounds 6, 9-10,21-24, 26, 28, 31-32, and 52-55, and are incorporated herein byreference.

EXAMPLE 84 ##STR6## SLB506

This exemplary pipecolic acid derivative is disclosed by Teague et al.,Bioorganic & Med. Chem. Letters, Vol. 4, No. 13, pp. 1581-1584, 1994,and is incorporated herein by reference.

EXAMPLES 85-88

Exemplary pipecolic acid derivative compounds are disclosed by Stocks etal., Bioorganic & Med. Chem. Letters, Vol. 4, No. 12, pp. 1457-1460,1994, particularly compounds 2, 15-17 and are incorporated herein byreference.

EXAMPLES 90-111

Additional exemplary pipecolic acid derivatives are described in Scheme10, Tables 1-5.

    ______________________________________                                        SCHEME 01                                                                      ##STR7##                                                                     EXAMPLE/COMPOUND      STRUCTURE                                               ______________________________________                                        6                     X = H.sub.2                                             7                     X = CH.sub.2                                            8                     X = H, CH.sub.3                                         9                     X = O                                                   ______________________________________                                    

    ______________________________________                                        SCHEME 2                                                                       ##STR8##                                                                     EXAMPLE/                                                                      COMPOUNDS No. R.sub.2                                                         ______________________________________                                        11                                                                                           ##STR9##                                                       12                                                                                           ##STR10##                                                      13                                                                                           ##STR11##                                                      14                                                                                           ##STR12##                                                      15                                                                                           ##STR13##                                                      16                                                                                           ##STR14##                                                      17                                                                                           ##STR15##                                                      18                                                                                           ##STR16##                                                      ______________________________________                                    

    ______________________________________                                        SCHEME 3                                                                      EXAMPLE/                                                                      COMPOUND NO.                                                                             STRUCTURE                                                          ______________________________________                                        19                                                                                        ##STR17##                                                         20                                                                                        ##STR18##                                                         21                                                                                        ##STR19##                                                         ______________________________________                                    

                  TABLE 1                                                         ______________________________________                                        Scheme 4                                                                       ##STR20##                                                                    Example/Compound No.                                                                            Structure                                                   ______________________________________                                        22                y = 1                                                       23                y = 2                                                       24                y = 3                                                       ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                         ##STR21##                                                                    Example/Compound No.                                                                            Structure                                                   ______________________________________                                        25                n = 1                                                       26                n = 2                                                       27                n = 3                                                       ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                         ##STR22##                                                                    Example/Compound No.                                                                            Structure                                                   ______________________________________                                        28                n = 1                                                       29                n = 2                                                       30                n = 3                                                       ______________________________________                                    

    ______________________________________                                        SCHEME 5                                                                       ##STR23##                                                                    EXAMPLE/                                                                      COMPOUND No. STRUCTURE                                                        ______________________________________                                        32           R = phenyl                                                       33           R = N(allyl).sub.2                                               34                                                                                          ##STR24##                                                       ______________________________________                                    

                  TABLE 1                                                         ______________________________________                                        Scheme 6                                                                       ##STR25##                                                                                        Structure                                                 Example/Compound No.                                                                              R                                                         ______________________________________                                        35                                                                                                 ##STR26##                                                36                                                                                                 ##STR27##                                                37                                                                                                 ##STR28##                                                38                                                                                                 ##STR29##                                                39                                                                                                 ##STR30##                                                40                                                                                                 ##STR31##                                                41                                                                                                 ##STR32##                                                42                                                                                                 ##STR33##                                                43                                                                                                 ##STR34##                                                44                                                                                                 ##STR35##                                                45                                                                                                 ##STR36##                                                46                                                                                                 ##STR37##                                                47                                                                                                 ##STR38##                                                48                                                                                                 ##STR39##                                                49                                                                                                 ##STR40##                                                50                                                                                                 ##STR41##                                                ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________    Example/Compound No.                                                                     Structure                                                          __________________________________________________________________________    51                                                                                        ##STR42##                                                         52                                                                                        ##STR43##                                                         53                                                                                        ##STR44##                                                         54                                                                                        ##STR45##                                                         55                                                                                        ##STR46##                                                         __________________________________________________________________________

                  TABLE 1                                                         ______________________________________                                        SCHEME 7                                                                       ##STR47##                                                                    ______________________________________                                        56           x = OH                                                           57           x = OMe                                                          58           x = Oi Pr                                                        59           x = OBn                                                          60           x = OCH MePh                                                     61           x = OCH.sub.2 CHCHPh                                             62           x = OCH.sub.2 CH.sub.2 CH.sub.2 (3,4-OMe.sub.2)Ph                63           x = NHBn                                                         64           x = NHCH.sub.2 CH.sub.2 CH.sub.2 Ph                              ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                         ##STR48##                                                                    ______________________________________                                        65                   R = Me                                                   66                   R = Bn                                                   ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        67                                                                                    ##STR49##                                                             68                                                                                    ##STR50##                                                             ______________________________________                                    

                                      TABLE 1                                     __________________________________________________________________________    Scheme 8                                                                       ##STR51##                                                                    Example/                                                                      Compound No.                                                                          Structure                                                             __________________________________________________________________________    69                                                                                     ##STR52##                                                                    R.sub.2 = Phe-o-tert-butyl                                            70                                                                                     ##STR53##                                                                    R.sub.1 = Phe-o-tert-butyl                                            __________________________________________________________________________

                  TABLE 2                                                         ______________________________________                                         ##STR54##                                                                    ______________________________________                                        71        R.sub.1 = m-OCH.sub.3 Ph; R.sub.3.sup.1 = Val-o-tert-butyl          72        R.sub.1 = m-OCH.sub.3 Ph; R.sub.3.sup.1 = Leu-o-tert-butyl          73        R.sub.1 = m-OCH.sub.3 Ph; R.sub.3.sup.1 = Ileu-o-tert-butyl         74        R.sub.1 = m-OCH.sub.3 Ph; R.sub.3.sup.1 = hexahydro-Phe-o-tert-               1                                                                             butyl                                                               75        R.sub.1 = m-OCH.sub.3 Ph; R.sub.3.sup.1 = allylalanine-o-tert-                butyl                                                               76        R.sub.1 = B-naphthyl; R.sub.3.sup.1 = Val-o-tert-butyl              ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                         ##STR55##                                                                    Example/Compound No.                                                                            Structure                                                   ______________________________________                                        77                R.sub.1 = CH.sub.2 (CO)-m-OCH.sub.3 PH                                        R.sub.4.sup.1 = CH.sub.2 Ph                                                   R.sub.5.sup.1 = OCH.sub.3                                   78                R.sub.1 = OH.sub.2 (CO)-B-naphthyl                                            R.sub.4.sup.1 = CH.sub.2 Ph                                                   R.sub.5.sup.1 = OCH.sub.3                                   ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                         ##STR56##                                                                    Example/Compound No.                                                                              Structure                                                 ______________________________________                                        79                  R.sub.1 = m-OCH.sub.3 Ph                                                      X = trans-CHCH                                                                R.sub.4.sup.1 = H                                                             Y = OC(o)Ph                                               80                  R.sub.1 = m-OCH.sub.3 Ph                                                      X = trans-CHCH                                                                R.sub.4.sup.1 = H                                                             Y = OC(o)CF.sub.3                                         81                  R.sub.1 = m-OCH.sub.3 Ph                                                      X = trans-CHCHI                                                               R.sub.4.sup.1 = --                                                            Y = --                                                    82                  R.sub.1 = m-OCH.sub.3 Ph                                                      X = trans-CHCH                                                                R.sub.4.sup.1 = H                                                             Y = OCH.sub.2 CHCH.sub.2                                  83                  R.sub.1 = m-OCH.sub.3 Ph                                                      X = C = O                                                                     R.sub.4.sup.1 = H                                                             Y = Ph                                                    ______________________________________                                    

                  TABLE 1                                                         ______________________________________                                        Scheme 9                                                                      85                                                                             ##STR57##                                                                    ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                         ##STR58##                                                                    ______________________________________                                        86            R.sub.1 = H, R.sub.2 = OMe R.sub.3 = CH.sub.2 OMe               87            R.sub.1 = H, R.sub.2 = R.sub.3 = H                              88            R.sub.1 = Me, R.sub.2 = R.sub.3 = H                             ______________________________________                                    

                  TABLE 1                                                         ______________________________________                                        Scheme 10                                                                      ##STR59##                                                                    Example           R =                                                         ______________________________________                                        90                3,4-dichloro                                                91                3,4,5-trimethoxy                                            92                H                                                           93                3-(2,5-Dimethoxy)-                                                            phenylpropyl                                                94                3-(3,4-Methylene-                                                             dioxy)phenylpropyl                                          ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                         ##STR60##                                                                    Example           R =                                                         ______________________________________                                        95                4-(p-Methoxy)-butyl                                         96                3-Phenylpropyl                                              97                3-(3-Pyridyl)-                                                                propyl                                                      ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                         ##STR61##                                                                    Example      R =                                                              ______________________________________                                         98          3-(3-Pyridyl)-propyl                                              99          1,7-Diphenyl-4-heptyl                                            100          4-(4-Methoxy)butyl                                               101          1-Phenyl-6-(4-methoxy-                                                        phenyl)-4-hexyl                                                  102          3-(2,5-Dimethoxy)phenyl-                                                      propyl                                                           103          3-(3,4-Methylenedioxy)-                                                       phenylpropyl                                                     104          1,5-Diphenylpentyl                                               ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                         ##STR62##                                                                    Example           R =                                                         ______________________________________                                        105               4-(4-Methoxy)butyl                                          106               3-Cyclohexylpropyl                                          107               3-Phenylpropyl                                              ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                         ##STR63##                                                                    Example           R =                                                         ______________________________________                                        108               3-Cyclohexylpropyl                                          109               3-Phenylpropyl                                              110               4-(4-Methoxy)butyl                                          111               1,7-Diphenyl-4-                                                               heptyl                                                      ______________________________________                                    

In addition to the chemical formulas provided above, the chemical namesfor compounds 90-111 are also provided below:

Compound No. Name of Species

90 (E)-3-(3,4-dichlorophenyl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate

Compound No. Name of Species

91 (E)-3-(3,4,5-trimethoxyphenyl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate

92 (E)-3-phenyl-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate

93 (E)-3-((3-(2,5-dimethoxy)-phenylpropyl)phenyl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate

94 (E)-3-(1,3-benzodioxol-5-yl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate

95 4-(4-methoxyphenyl)butyl1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate

96 3-phenylpropyl 1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate

97 3-(3-pyridyl)propyl 1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate

98 3-(3-pyridyl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate

99 4-phenyl-1-(3-phenylpropyl)butyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate

100 4-(4-methoxyphenyl)butyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate

101 1-(4-methoxyphenethyl)-4-phenylbutyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate

102 3-(2,5-dimethoxyphenyl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate

103 3-(1,3-benzodioxol-5-yl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate

Compound No. Name of Species

104 1-phenethyl-3-phenylpropyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate

105 4-(4-methoxyphenyl)butyl1-(2-cyclohexyl-2-oxoacetyl)-2-piperidinecarboxylate

106 3-cyclohexylpropyl1-(2-cyclohexyl-2-oxoacetyl)-2-piperidinecarboxylate

107 3-phenylpropyl 1-(2-cyclohexyl-2-oxoacetyl)-2-piperidinecarboxylate

108 3-cyclohexylpropyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate

109 3-phenylpropyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate

110 4-(4-methoxyphenyl)butyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate

111 4-phenyl-1-(3-phenylpropyl)butyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate

NEUROTROPHIC EFFECTS OF ROTAMASE INHIBITORS

Table I lists a number of the claimed examples together with theirpotencies to induce trophic effects in cultured sensory neurons, asdescribed above. FIGS. 19 and 20 show photomicrographs of Example 111and Example 17 promoting neurite outgrowth in the dorsal root ganglioncultures.

                  TABLE I                                                         ______________________________________                                        In Vitro Potencies of Test Examples                                                       Rotamase Inhibition                                                                        Neutrophic ED.sub.50                                 Example     K.sub.i, nM  Chick DRGs, nM                                       ______________________________________                                        6           140          25                                                   9           13           0.030                                                11          170          1                                                    12          250          300                                                  13          25           80                                                   15          17           0.30                                                 19          12           0.017                                                36          >10,000      >10,000                                              41          1300         5000                                                 50          >10,000      >10,000                                              90          1800         2500                                                 91          28           200                                                  92          39           90                                                   93          75           35                                                   94          70           8                                                    95          165           5-10                                                96          740          10-20                                                97          725          150                                                  98          130          75                                                   99          30           5                                                    100         60           43                                                   101         15           0.17                                                 102         12           2.5                                                  103         120          3                                                    104         20           .016                                                 105         103          6                                                    106         760          1                                                    107         210          0.82                                                 108         32           0.29                                                 109         2            0.08                                                 110         24           0.002                                                111         5            0.08                                                 ______________________________________                                    

ACTIVITY OF EXAMPLE COMPOUNDS IN IN VIVO MODEL OF NERVE REGENERATION

Sciatic Nerve Axotomy

Six-week old male Sprague-Dawley rats were anesthetized, and the sciaticnerve exposed and crushed, at the level of the hip, by forceps. Testcompounds or vehicle were administered subcutaneously just prior to thelesion and daily for the following 18 days. Sections of the sciaticnerve were stained with Holmes silver stain to quantify the number ofaxons, and Luxol fast blue to quantify the level of myelination.Eighteen days after lesion, there was a significant decrease in thenumber of axons (50% decrease as compared to non-lesioned control) anddegree of myelination (90% decrease as compared to non-lesioned control)in animal treated with vehicle.

Administration of Example 12 (30 mg/kg s.c.), or Example 13 (mg/kg s.c.)just prior to the lesion and daily for 18 days following the lesion,resulted in significant regeneration of both axon number (25% and 5%decrease, respectively, as compared to non-lesioned control) and thedegree of myelination (65% and 50% decrease, respectively, as comparedto control) as compared to vehicle treated animals. The significantefficacy of Examples 12 and 13 are consistent with their potent activityin inhibiting rotamase activity and stimulating neurite outgrowth inchick DRGs, and their relative potencies in vivo parallel their in vitropotencies (Table I). These results are shown in FIG. 21. "Sham" denotescontrol animals that received vehicle but were not lesioned; "Vehicle"denotes animals that were lesioned and received only vehicle (i.e., nodrug). Example 12 and Example 13 showed a striking similarity to thesham treated animals, demonstrating the powerful neuroregenerativeeffects of these compounds in vivo. These data are quantitated in TableII.

                  TABLE II                                                        ______________________________________                                                      Axon Number                                                     Treatment     (% Control)                                                                              Myelin Level                                         ______________________________________                                        Sham          100        100                                                  Lesion:                                                                       + Vehicle (s.c.)                                                                            50         10                                                   + Example 12  75         35                                                   (30 mg/kg s.c.)                                                               + Example 13  100        50                                                   (30 mg/kg s.c.)                                                               ______________________________________                                    

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention and all suchmodifications are intended to be included within the scope of thefollowing claims.

What is claimed is:
 1. A method of treating a neurological disorder inan animal, comprising:administering to an animal an effective amount ofa non-immunosuppressive pipecolic acid derivative having an affinity forFKBP-type immunophilins to stimulate growth of damaged peripheral nervesor to promote neuronal regeneration, wherein the FKBP-type immunophilinexhibits rotamase activity and the pipecolic acid derivative inhibitssaid rotamase activity of the immunophilin, wherein saidnon-immunosuppressive pipecolic derivative is selected from the groupconsisting of:(E)-3-(3,4-dichlorophenyl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-(3,4,5-trimethoxyphenyl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-phenyl-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-((3-(2,5-dimethoxy)-phenylpropyl)phenyl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-(1,3-benzodioxol-5-yl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate; 3-phenylpropyl1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate; 3-(3-pyridyl)propyl1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate; 3-(3-pyridyl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-phenyl-1-(3-phenylpropyl)butyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;1- (4-methoxyphenethyl)-4-phenylbutyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;3-(2,5-dimethoxyphenyl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;3-(1,3-benzodioxol-5-yl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;1-phenethyl-3-phenylpropyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(2-cyclohexyl-2-oxoacetyl)-2-piperidinecarboxylate; 3-cyclohexylpropyl1-(2-cyclohexyl-2-oxoacetyl1-2-piperidinecarboxylate; 3-phenylpropyl1-(2-cyclohexyl-2-oxoacetyl)-2-piperidinecarboxylate; 3-cyclohexylpropyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate; 3-phenylpropyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate; and4-phenyl-1-(3-phenylpropyl)butyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate.2. The method of claim 1, wherein the neurological disorder is selectedfrom the group consisting of peripheral neuropathies cause by physicalinjury or disease state, physical damage to the brain, physical damageto the spinal cord, stroke associated with brain damage, andneurological disorders relating to neurodegeneration.
 3. The method ofclaim 2, wherein the neurological disorder is selected from the groupconsisting of Alzheimer's Disease, Parkinson's Disease, and amyotrophiclateral sclerosis.
 4. The method of claim 1 wherein thenon-immunosuppressive pipecolic acid derivative is administered orally.5. A method of treating a neurological disorder in an animal,comprising:administering to an animal an effective amount of anon-immunosuppressive pipecolic acid derivative having an affinity forFKBP-type immunophilins in combination with an effective amount of aneurotrophic factor selected from the group consisting of neurotrophicgrowth factor, brain derived growth factor, glial derived growth factor,cilial neurotrophic factor, and neurotropin-3, to stimulate growth ofdamaged peripheral nerves or to promote neuronal regeneration, whereinthe FKBP-type immunophilin exhibits rotamase activity and the pipecolicacid derivative inhibits said rotamase activity of the immunophilin,wherein said non-immunosuppressive pipecolic derivative is selected fromthe group consisting of:(E)-3-(3,4-dichlorophenyl)-2-propenyl1-3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-(3,4,5-trimethoxyphenyl)-2-propenyl1-3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-phenyl-2-propenyl1-3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-((3-(2,5-dimethoxy)-phenylpropyl)phenyl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-(1,3-benzodioxol-5-yl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate; 3-phenylpropyl1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate; 3-(3-pyridyl)propyl1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate; 3-(3-pyridyl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-phenyl-1-(3-phenylpropyl)butyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;1-(4-methoxyphenethyl)-4-phenylbutyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;3-(2,5-dimethoxyphenyl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;3-(1,3-benzodioxol-5-yl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;1-phenethyl-3-phenylpropyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(2-cyclohexyl-2-oxoacetyl)-2-piperidinecarboxylate; 3-cyclohexylpropyl1-(2-cyclohexyl-2-oxoacetyl)-2-piperidinecarboxylate; 3-phenylpropyl1-(2-cyclohexyl-2-oxoacetyl)-2-piperidinecarboxylate; 3-cyclohexylpropyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate; 3-phenylpropyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl 1-(3,3-dimethyl-2-oxobutanoyl )-2-piperidinecarboxylate; and4-phenyl-1-(3-phenylpropyl)butyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate.6. The method of claim 5, wherein the neurological disorder is selectedfrom the group consisting of peripheral neuropathies cause by physicalinjury or disease state, physical damage to the brain, physical damageto the spinal cord, stroke associated with brain damage, andneurological disorders relating to neurodegeneration.
 7. The method ofclaim 6, wherein the neurological disorder is selected from the groupconsisting of Alzheimer's Disease, Parkinson's Disease, and amyotrophiclateral sclerosis.
 8. The method of claim 5 wherein thenon-immunosuppressive pipecolic acid derivative is administered orally.9. A method of stimulating growth of damaged peripheral nerves,comprising:administering to damaged peripheral nerves an effectiveamount of a non-immunosuppressive pipecolic acid derivative compoundhaving an affinity for FKBP-type immunophilins to stimulate or promotegrowth of the damaged peripheral nerves, wherein the FKBP-typeimmunophilins exhibit rotamase activity and the pipecolic acidderivative inhibits said rotamase activity of the immunophilin, whereinsaid non-immunosuppressive pipecolic derivative is selected from thegroup consisting of:(E)-3-(3,4-dichlorophenyl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-(3,4,5-trimethoxyphenyl)-2-propenyl-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-phenyl-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-((3-(2,5-dimethoxy)-phenylpropyl)phenyl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-(1,3-benzodioxol-5-yl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate; 3-phenylpropyl1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate; 3-(3-pyridyl)propyl1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate; 3-(3-pyridyl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-phenyl-1-(3-phenylpropyl)butyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;1-(4-methoxyphenethyl)-4-phenylbutyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;3-(2,5-dimethoxyphenyl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;3-(1,3-benzodioxol-5-yl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;1-phenethyl-3-phenylpropyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(2-cyclohexyl-2-oxoacetyl)-2-piperidinecarboxylate; 3-cyclohexylpropyl1-(2-cyclohexyl-2-oxoacetyl ) -2-piperidinecarboxylate; 3-phenylpropyl1-(2-cyclohexyl-2-oxoacetyl)-2-piperidinecarboxylate; 3-cyclohexylpropyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate; 3-phenylpropyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate; and4-phenyl-1-(3-phenylpropyl)butyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate.10. The method of claim 9, further comprising administering aneurotrophic factor to stimulate or promote growth of the damagedperipheral nerves selected from the group consisting of neurotrophicgrowth factor, brain derived growth factor, glial derived growth factor,cilial neurotrophic factor, and neurotropin-3.
 11. The method of claim 9wherein the non-immunosuppressive pipecolic acid derivative isadministered orally.
 12. A method for promoting neuronal regenerationand growth in animals, comprising:administering to an animal aneffective amount of a non-immunosuppressive pipecolic acid derivativecompound having an affinity for FKBP-type immunophilins to promoteneuronal regeneration, wherein the FKBP-type immunophilins exhibitrotamase activity and the pipecolic acid derivative inhibits saidrotamase activity of the immunophilin, wherein saidnon-immunosuppressive pipecolic derivative is selected from the groupconsisting of:(E)-3-(3,4-dichlorophenyl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-(3,4,5-trimethoxyphenyl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-phenyl-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-((3-(2,5-dimethoxy)-phenylpropyl)phenyl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;(E)-3-(1,3-benzodioxol-5-yl)-2-propenyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate; 3-phenylpropyl1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate; 3-(3-pyridyl)propyl1-(2-oxo-2-phenylacetyl)-2-piperidinecarboxylate; 3-(3-pyridyl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-phenyl-1-(3-phenylpropyl)butyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;1-(4-methoxyphenethyl)-4-phenylbutyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;3-(2,5-dimethoxyphenyl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;3-(1,3-benzodioxol-5-yl)propyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;1-phenethyl-3-phenylpropyl1-(3,3-dimethyl-2-oxopentanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(2-cyclohexyl-2-oxoacetyl)-2-piperidinecarboxylate; 3-cyclohexylpropyl1-(2-cyclohexyl-2-oxoacetyl)-2-piperidinecarboxylate; 3-phenylpropyl1-(2-cyclohexyl-2-oxoacetyl)-2-piperidinecarboxylate; 3-cyclohexylpropyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate; 3-phenylpropyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate;4-(4-methoxyphenyl)butyl1-(3,3-dimethyl-2-oxobutanoyl)-2-piperidinecarboxylate; and4-phenyl-1-(3-Phenylpropyl)butyl1-(3,3-dimethyl-2-oxobutanoyl-2-piperidinecarboxylate.13. The method of claim 12, further comprising administering aneffective amount of a neurotrophic factor to promote neuronalregeneration selected from the group consisting of neurotrophic growthfactor, brain derived growth factor, glial derived growth factor, andneurotropin-3.
 14. The method of claim 12 wherein thenon-immunosuppressive pipecolic acid derivative is administered orally.